Hypersensitivity of cells from patients with Fanconi anemia (FA) to the clastogenic and cytotoxic effects of DNA interstrand cross-linking agents and their defect in ability to repair damage produced by these agents has led to the hypothesis that the etiopathogenesis of this disorder involves a DNA repair defect. The goals of this proposal are to delineate the relationship between the FANC proteins and those proteins involved in the critical, initial damage recognition and incision steps of the repair process and to ascertain their functional importance. We have identified a structural protein, nonerythroid a spectrin (aSpllS*) as a component of a protein complex in the nucleus of normal cells and shown that it binds to cross-linked DNA and is deficient in all FA cell lines tested. This deficiency is corrected in FA-A, FA-C and FA-G cells expressing the appropriate FANC cDNAs, indicating involvement of the FANC proteins. We have hypothesized that aSpllS* is a critical factor in the repair defect in FA cells, that it acts as a scaffold to help recruit repair proteins at sites of damage, aiding in their alignment and interactions, and that the interaction of aSpllS* with the FANC proteins is essential for the repair process. To address this hypothesis, yeast-two-hybrid analysis will be used to determine whether there are direct interactions between aSpllS*, the FANC proteins and DNA repair proteins involved in cross-link repair and ascertain the domains involved in these interactions. Whether the FANC and DNA repair proteins co-localize with aSpllS* at sites of damage-induced foci in the nucleus will be examined, as will the kinetics of this co-localization and the mechanism of localization of aSpllS* to these sites. Using siRNA-mediated silencing of aSpllS* and FANC gene expression in normal cells, the functional importance of these genes in the repair process will be ascertained. Also, whether the FANC proteins bind directly to cross-linked DNA will be determined and, if so, the specific binding domains on these proteins and on aSpllS* will be examined. These studies should elucidate the role of aSpllS* and the FANC proteins in repair and the importance of the interaction of aSpllS* with the FANC proteins for its stability. Since aII spectrin has been associated with a number of different processes in the cell besides DNA repair, such as signal transduction and cell growth and differentiation, a deficiency in aSpllS* in FA cells could have far reaching consequences. Thus elucidating the relationship between aSpllS* and the FANC proteins could potentially delineate the basis for defective hematopoietic differentiation and development, and for aplastic anemia, leukemia and other cancers in FA. [unreadable] [unreadable] [unreadable]